Bladder cancer kills 13,000 Americans each year, but very few research projects target this disease. Most of these deaths are due to metastatic spread, commonly to the lungs. A fuller understanding of the molecular mechanisms driving growth and dissemination of bladder cancer is likely to provide new therapeutic opportunities. RalA and RalB, two homologous GTPases play an important role in growth and lung metastasis in xenograft models of human bladder cancer. Furthermore, these paralogs are activated by phosphorylation at sites regulated by Aurora-A and PKC, two kinases associated with human bladder cancer growth and progression. To define signaling pathways downstream of Ral, genome-wide analysis of Ral dependent gene expression was carried out and revealed that RalA and RalB regulate the expression of CD24, a GPI-linked glycoprotein and biomarker of metastasis development in bladder cancer. Moreover, this data identified the zinc finger transcription factor RREB1 as a putative regulator of CD24 expression and RalBP1, an effector of both Ral paralogs, as a regulator of RREB1 activity. Based on this data, we formulate the Guiding Hypothesis that RalA and RalB regulate a new downstream signaling pathway consisting of RalBP1, RREB1 and CD24 via which they contribute to bladder cancer growth and lung metastasis and furthermore, that biomarkers of RalA and RalB activation such as phosphorylation or transcriptional signatures are prognostic for development of bladder cancer metastasis in patients. Three Specific Aims test this hypothesis: In Aim 1, the role of RalA and RalB phosphorylation in bladder cancer growth, migration and lung metastasis will be determined using phospho inactive mutants. The prognostic relevance of Ral paralog phosphorylation to metastasis development will be evaluated in tissues from patients with locally advanced bladder cancer. Aim 2 will determine how RalBP1 regulates RREB1 activity and how RREB1 in turn, regulates CD24 expression. Since genome-wide profiling of Ral dependent gene expression led to the discovery of CD24, we will use tissues from Aim 1 and advanced computational tools to generate paralog specific signatures of Ral expression. The ability of these signatures to predict the development of metastatic disease in patients will be evaluated. In Aim 3 we evaluate the requirement of CD24 for bladder carcinogenesis and progression. A CD24 knockout mouse will be used to study the necessity of CD24 in chemically induced bladder carcinogenesis and/or subsequent invasion and metastasis of resultant tumors. A separate study will test whether anti-CD24 antibodies can inhibit growth of established bladder cancer lung metastases. Establishing the biological relevance of Ral phosphorylation to bladder cancer lung metastasis and the regulatory connections between RalBP1, RREB1 and CD24 provide opportunities for therapy. Translational studies associated with this mechanistic work provide biomarker predictors for metastasis development in patients while a preclinical study provides a foundation for the near term development of novel therapies directed at established metastatic disease. PUBLIC HEALTH RELEVANCE: Bladder cancer kills 13,000 Americans each year, but few research projects are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. The goal of this project is to understand the mechanisms that underlie lung metastasis in human bladder cancer and use this knowledge to predict and treat this condition in patients.